Our combined in vitro biochemical and structural studies on a representative member of the IS200/IS605 transposase family demonstrated that this family uses a completely novel recombination pathway involving the movement of only single-stranded DNA. One particularly surprising discovery was that the transposase recognizes its target site through DNA-DNA interactions rather than using a site-specific DNA binding domain: target site recognition is accomplished by base pairing interactions between the target site and an internal segment of transposon DNA. This suggests the possibility that by changing the internal segment, targeting could be directed to novel target sites. If we can do this, this might allow the precise introduction of exogenous genes into benign locations in chromosomes or places where gene expression can be appropriately controlled in a cell- and development-specific manner. In our recent work, we have been exploring two aspects of IS200/IS605 transposition. The first is directed towards the intriguing question of how transposition occurs in cells since both the transposon and its target must be single-stranded yet most DNA in cells is double-stranded. We have used a variety of genetic and in vivo experiments to show that IS608 transposition is closely linked to the availability of ssDNA on the lagging strand at the replication fork. The other aspect we have been investigating is to determine if features of IS608 transposition are generalizable to the entire superfamily, by expanding our studies to another member of the IS200 superfamily, ISDra2. ISDra2 is particularly interesting as its transposition is specifically induced in Deinococcus radiodurans upon UV or gamma irradiation (Mennecier et al., 2006). We have solved a series of ISDra2 transposase-DNA complexes, and shown that although many of the principles of targeting found for IS608 are applicable, there are mechanistically important differences. One noteworthy difference is that ISDra2 recognizes five nucleotides at its target site, rather than the four of IS608. The manner in which ISDra2 accomplishes this extends our understanding of the mechanism of transposition. We have also been able to structurally capture the pre-cleavage state along the pathway, a snapshot that eluded us with the IS608 system. In other related work, we have also solved the structure of a related HUH nuclease that binds and processes short Repeated Extragenic Palindromic Sequences (REPs) in bacteria, also in complex with a DNA palindrome. Curcio, M.J. and Derbyshire, K.M. (2003) Nat. Rev. Mol. Cell. Biol. 4, 865-877. Debets-Ossenkopp, Y.J., et al. (1999) Antimicrob. Agents Chemother. 43, 2657-2662. Kersulyte, D., et al. (2002) J. Bacteriol. 184, 992-1002. Mennecier, S., Servant, P., Coste, G., Bailone, A., and Sommer, S. (2006) Mol. Microbiol. 59, 317-325. Sebaihia, M. et al. (2006) Nature Genet. 38, 779-786.